Many industries need to detect and quantify the concentration and level of biological material in a liquid sample. For example, the determination of bacterial concentration in water is an essential part of water quality testing. EPA regulations require that no Coliform or Escherichia coli can be present in potable water. The "presence/absence" format of a testing medium, such as COLILERT.RTM. or COLILERT-18.TM. chemical mixture (IDEXX Laboratories, ME), either of which is used as a testing medium for Escherichia coli and coliform bacteria, is very useful in making this determination. COLILERT.RTM. chemical mixture is based on the Defined Substrate Technology described in Edberg, "Method and Medium for use in Detecting Target Microbes In Situ in A Specimen Sample of A Possibly Contaminated Material," U.S. Pat. No. 4,925,789.
However, there are areas where the quantification, not just the detection, of bacterial concentration is important. Examples of such areas include water waste, incoming water in water purification systems, surface water, and food testing.
The classical methods of quantification of biological material are membrane filtration and the most probable number method.
In membrane filtration, the required volume of sample is filtered through a membrane of a very small pore size to non-specifically trap bacteria. The membrane is then placed on a medium which supports the growth of the target bacteria. The medium is then incubated at a specific temperature for a specific time, and any resulting colonies are counted. A drawback of membrane filtration is that a sample which contains particles other than bacteria (e.g., a waste water sample) may clog the membrane and make it unusable. Another drawback is that the membrane traps non-target bacteria.
The most probable number method is described in Recles et al., "Most Probable Number Techniques" published in "Compendium of Methods for the Microbiological Examination of Foods", 3rd ed. 1992, at pages 105-199, and in Greenberg et al., "Standard Methods For the Examination of Water and Wastewater" (8th ed. 1992). In this method, a volume of water sample is dispensed into several tubes (e.g., 10.times.10, 10 tubes each containing 10 ml) and bacteria in each tube allowed to grow. After incubation at a specific temperature for a specific time, the number of positive tubes is counted. The most probable number can be determined from the formula: ##EQU1## where p is the number of positive tubes, N is the ml sample in negative tubes, T is the ml sample in all tubes, and MPN is the most probable number. A major drawback of the method is the range of 95% confidence limits is large, when only a few tubes are used. Such confidence limits are calculated roughly as: ##EQU2## where n is the number of tests.